Abstract 5054: Understanding the genetic heterogeneity within clonal spheroids and its interaction with the tumor microenvironment

Introduction: Head and neck squamous cell carcinomas (HNSCC) exhibit significant inter- and intra-tumor heterogeneity, complicating diagnosis and treatment. Our previous work demonstrated that spheroids developed from patient-derived cells and HNSCC cell lines display distinct proliferative and migratory behaviors. We classified them as hyperproliferative and hypoproliferative spheriods. To uncover the molecular basis of this functional heterogeneity, we utilized single-cell RNA sequencing (scRNA-seq) and advanced computational tools like CellChat. Our analysis revealed key signaling pathways, cellular interactions, and surface markers defining spheroid phenotypes, offering insights into their genetic and functional diversity. Material and Methods: scRNA-seq was performed on hyperproliferative and hypoproliferative spheroids derived from the UMSCC-22B cell line. Spheroids were cultured, dissociated into single cells, and subjected to high-throughput scRNA-seq. Bioinformatic analyses identified differentially expressed genes, signaling pathways, and cellular states. CellChat was employed to analyze intercellular communication networks within spheroids. Ligand-receptor interactions were modelled to identify pathways mediating cross-talk between distinct cell clusters. Results: scRNA-seq revealed distinct transcriptional profiles between the two spheroids. Hyperproliferative spheroids expressed genes related to cell division and growth, while hypoproliferative spheroids expressed genes linked to migration and invasion. Pathway analysis identified differences in angiogenesis, metastasis, and tumor microenvironment interactions. Midkine signalling(MDK-SDC) through basal cells was found to be the principal contributor to the proliferative advantage in hyperproliferative spheriods, supporting their role in tumor expansion and progression. While, Desmosomal signaling (DSC2-DSG2) through keratinocytes and fibroblasts was found to play a critical role in maintaining cellular adhesion and promoting migratory capacity in hypoproliferative spheroids. TCGA data linked MDK to advanced tumor stages, poor survival, and therapy resistance, while DSG2 and DSC2 were overexpressed in metastatic tumors. These findings highlight the distinct functional states of spheroids contributing to tumor heterogeneity. Conclusion: The balance between proliferation and migration in spheroids reflects tumor heterogeneity's dual role in cancer progression. Midkine-driven hyperproliferative spheroids anchor tumor growth, while desmosome-mediated hypoproliferative spheroids drive metastasis. Targeting Midkine or modulating desmosomal remodeling could inhibit growth and prevent dissemination, enabling patient-specific therapies. Citation Format: Anshu Yadav, Rupesh Chaturvedi, Rana P. Singh. Understanding the genetic heterogeneity within clonal spheroids and its interaction with the tumor microenvironment [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 5054.